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Abstract

The technology of whole image acquisition from histological glass slides (Virtual
slides, (VS)) and its associated software such as image storage, viewers, and virtual
microscopy (VM), has matured in the recent years. There is an ongoing discussion whether
to introduce VM into routine diagnostic surgical pathology (tissue-based diagnosis)
or not, and if these are to be introduced how best to do this. The discussion also
centres around how to substantially define the mandatory standards and working conditions
related to introducing VM. This article briefly describes some hypotheses alongside
our perspective and that of several of our European colleagues who have experienced
VS and VM either in research or routine praxis. After consideration of the different
opinions and published data the following statements can be derived: 1. Experiences
from static and remote telepathology as well as from daily routine diagnoses, confirm
that VM is a diagnostic tool that can be handled with the same diagnostic accuracy
as conventional microscopy; at least no statistically significant differences (p > 0.05) exist. 2. VM possesses several practical advantages in comparison to conventional
microscopy; such as digital image storage and retrieval and contemporary display of
multiple images (acquired from different stains, and/or different cases). 3. VM enables
fast and efficient feedback between the pathologist and the laboratory in terms of
ordered additional stains, automated access to the latest research for references,
and fast consultation with outstanding telepathology experts. 4. Industry has already
invested “big money” into this technology which certainly will be of influence in
its future development. The main constraints against VM include the questionable reimbursement
of the initial investment, the missing direct and short term financial benefit, and
the loss of potential biological identity between the patient and the examined tissue.
This article tries to analyze and evaluate the factors that influence the implementation
of VM into routine tissue-based diagnosis, for example in combination with predictive
diagnosis. It focuses on describing the advantages of modern and innovative electronically
based communication technology.

Virtual Slides

Introduction

Tissue based diagnosis derived from human tissue(s) still remains the most reliable
method of disease recognition and classification in both sensitivity and specificity
[1]. For example, in the USA only 272 surgical pathology claims from 1998–2003 have been
reported to have significant therapeutic consequences and an inter-observer error
rate of 80–95% dependent upon technical procedure (fine needle aspiration, etc.),
and diagnosis (Spitz nevus versus melanoma, etc.) [2,3]. All these studies rely on manual workflow and manual tissue processing. Considerations
on the impact of digitised tissues or slides, and electronic communication in terms
of expert consultation, have not been included up until now; to our knowledge.

The new technology of image acquisition and digitisation (virtual slides, (VS)) has
been broadly applied in nearly all Institutes of Pathology related to Universities
in Germany and other European countries. It has been used for education, storage of
rare cases, clinical pathological conferences, and research assessment such as tissue
micro arrays (TMA) [4,5]. It’s implementation into the routine research workflow is still in its initial stages.
Some private institutes of pathology are already reporting satisfying and good results
when deriving diagnoses from routinely acquired VS [6-8].

Which standards and control functions need to be fulfilled by the corresponding laboratories
and technical equipment specialists in order to ascertain or corroborate the high
level of quality of diagnostic surgical pathology in VM?

Conventional laboratory workflow and tissue-based diagnosis

Human tissue needs to be fixed, processed, cut, stained, and finally analyzed by a
surgical pathologist. Specific equipment is commonly used for fixation, tissue processing,
and staining, which is loaded manually [9,10]. Microtome cuts usually require manual performance. The whole workflow is monitored
by a laboratory information system (LIS), which itself is subject to accreditation
and certification [11]. Capable LIS report the stages of individual tissues at any time and step of the
workflow [12,13]. The quality of the obtained slides varies. Slides of good quality allow easier and
more precise diagnoses; although experienced pathologists well versed in specific
laboratory parameters can view slides of poorer quality with similar diagnostic accuracy.
This statement, however, only holds true for “common H&E based” diagnoses, such as
tuberculosis or squamous cell carcinoma.

The demands of guiding an adequate therapy have led to the term “tissue–based diagnosis”
which includes H&E based diagnosis, prognosis associated diagnosis (usually demanding
immunohistochemical investigations (IHC)), predictive diagnosis (usually demanding
IHC and gene mutation analysis (PCR)), and risk estimation, which is the estimation
of risk prior to the establishment of a certain disease (usually demanding gene analysis
(PCR)) [14,15]. Prognosis associated diagnosis, predictive diagnosis, and risk estimation are more
sensitive to variations in technical performance in comparison to H&E based diagnosis.
They have to be continuously monitored and are sensitive to the fixation medium [16]. Derived from the ring investigations in clinical pathology, IHC standard stained
slides are distributed and judged by referees in several European countries (Germany,
Great Britain, and others) [17,18]. The diagnostic accuracy is controlled by distributing VS to surgical pathologists
and evaluating their responses via electronic communication [13,19].

When analyzing the laboratory workflow, a principal component has to be included:
the primary H&E diagnosis has to be confirmed and further refined by additional laboratory
procedures such as additional stains like PAS or Giemsa, etc., IHC or additional investigations
such as PCR, or in situ hybridization, etc. In a laboratory with a conventional workflow
they are ordered by the responsible pathologist and depend upon the demands of the
clinician. All these actions can be aggregated under the topic reliable “communication”
and user – friendly “man – machine interaction”. Herein, appropriate digital compartments
are essential.

Digital pathology and tissue-based diagnosis

The acquisition of virtual slides and associated virtual microscopy (VM) to interpret
digital images occur close to the end of the diagnostic chain, typically just before
the diagnosis is reported. It is useful to distinguish between interactive and automated
VM [20]. Interactive VM is the pathologist’s diagnostic performance when viewing VS. Automated
VM includes the implementation of computer-aided “diagnostic assistants” [21]. They can be compared with program assistants that assist in detecting spelling errors
in a letter, or in formatting certain documents, etc. [21,22]. Most vendors concentrate on interactive VM. Some add IHC measurement programs that
have been designed to calculate IHC scores such as Her2/neu or hormone receptor status
in breast cancer [23]. Outside of gynaecological cytopathology, automated VM has not been fully implemented
to the best of my knowledge.

Obviously, the quality of VS depends upon the original slides and upon the colour
and spatial accuracy of the whole slide imaging (WSI) scanner [13,23] which is used. Most WSI scanners can be equipped with objectives of different magnification
(i.e., x10, x20, and x40) which define the scanning time and mainly the number of
focal z-planes. In histology (cellular structure based morphology) one individual
plane is usually sufficient to render a diagnostic interpretation, in contrast to
cytology (intra-cellular structure based morphology) which requires several focal
planes (referring to the Z-axis) [13].

Implementing VM into routine practice offers several principle advantages, which can
be summarized as follows:

1. Once a scanner delivers VS of sufficient diagnostic quality, the technology generally
continues to produce images of similar quality. Working with a conventional microscope
requires frequent “resetting” of the focus plane, adjustment of brightness, etc. Conventional
microscope settings also depend upon personal preferences, which can be simulated
by VS viewers if desired.

2. Several images obtained from different stains of the same case or tumor can be
viewed simultaneously with VM. This feature allows for a more precise judgement of
prognosis associated diagnosis and predictive diagnosis.

3. Quantitative evaluation of object and structure features can be evaluated either
interactively or automatically, which is of assistance in prognosis associated diagnosis
and predictive diagnosis.

4. Annotations can be included for teaching, marking areas of interest, or discussion
purposes in VM.

The advantages of maintaining the conventional performance of tissue-based diagnosis,
which are identical with the constraints of the implementation of VM include:

1. The lack of financial advantage of VM implementation in conjunction with the
decreasing prices for hardware and software. This is in direct contrast to the financial
advantage of implementing digital imaging on live images in disciplines such as radiology,
which saves the expenses of paper based diagnosis (films). The investigations are
not negligible and can amount to about 20% of the gross budget dependent upon the
size of the pathology institute (private estimation). There seem to be no savings
when implementing VM, because the processes in the laboratory mainly remain untouched
and only the workflow is affected. Some authors however have mentioned the comparable
save in labour when retrieving a case and the increased safety of diagnoses using
VM [6,8]. However, these are only secondary savings, and mainly depend upon the local environment
[4,24].

2. Security of the patient’s diagnosis, and the possibility to control the origin
of the glass slides by finger printing the patient’s DNA at any time. Obviously, this
cannot be stated in VS, however, modern electronic safety controls permit an unambiguous
relationship between the VM and the original glass slides [25].

3. Additional arguments such as difficulties to view and diagnose VS compared to
conventional microscopes depend more or less on training and performance of the individual
pathologist [24,26,27]. This has also been demonstrated in teaching courses using VS and conventional microscopes
for medical students in anatomy [24,28-31]. If appropriately trained, most of the students in these courses preferred VS compared
to receiving training with conventional microscopes [12].

In summary, VM offers greater advantages than conventional light microscopy with respect
to prognosis associated diagnosis and predictive diagnosis, i.e., for those types
of diagnoses which are closely associated with guiding patient therapy.

Consecutives

Tissue-based diagnosis still remains related to the pathologist’s knowledge in both
conventional microscopy and VM. The diagnosis information sources include the patient’s
history, microscopic and gross images, and additional findings (live imaging, etc.).
Microscopic images including VS contribute significantly, however not in total, to
the patient’s diagnosis. The more distinct a diagnosis, the more information in addition
to microscopic images is required for an adequate diagnosis to be made [13,32-34].

The optical pathways are important to obtain good images, but are not the only prerequisite.
Preservation, fixation, and embedding of tissue, cutting of tissue blocks, and staining
of glass slides also contribute to VS quality to a high degree. From our experience
in Europe, the WSI scanner performance (i.e. the acquisition of VS) is of the highest
quality to permit diagnostic interpretations to be made.

What can we conclude?

1. The histological diagnosis is based not only on the final digital image, but
also upon a chain of laboratory procedures with additive external clinical information.

2. Diagnoses have to be delivered at different levels in relation to the clinical
demands and potential treatment needs of the patient.

3. “Crude diagnoses”, for example H&E diagnoses, are adequate in many situations,
for example in odontogenic cysts, acute appendicitis, common nevi, etc.

4. Adequate quality control of histological diagnoses should start with the final
outcome, i.e., analysing the diagnostic quality of the material being viewed in relation
to the demands of potential therapy.

5. The weakest link in the diagnostic chain should be addressed with high priority.
It can vary - for example, tissue preservation and fixation are subject to a broad
variance.

6. The optical pathway is the end link in the diagnostic workflow process, and therefore,
of less significance to associated laboratory reprocessing such as IHC, ISH, etc.

7. Monitoring intra-laboratory procedures (e.g., efficiency and reproducibility
of reprocessing and staining, etc.) should be separated from the evaluation of diagnosis
quality.

8. Evaluation of diagnostic errors should take into account the therapeutic consequences
and the local environment.

9. Automation of a diagnostic chain process is aimed at providing a consistent quality
of material for making diagnoses. Its variation should be analyzed with regard to
its influence on the final diagnosis.

10. The most efficient quality assurance in automated technology is the accuracy
afforded by quantification of measurement.

The emerging tendency to separate pre-diagnostic workflow steps in the histopathology
laboratory from the final stages that are important for pathologists to make diagnoses
are likely to create new quality assurance procedures in agreement with the above
listed statements. The contribution of VS to logistic and diagnostic inaccuracies
is probably less important when compared with tissue preservation and processing,
as all molecular biology and genetic investigations depend upon tissue preservation
and processing.

Many European pathologists can be conservative when working in a strictly regulated
financial environment. Financial issues play the main role in all non-research technological
or medical investments, and are strictly separated from research and scientific approaches.

In Germany, to my knowledge, several University Institutes of Pathology have already
undertaken new efforts to implement VM into routine diagnosis. Financial issues, in
particular the high investment and considerable expense of integrating VM into the
existing LIS, are hard to overcome due to restrictive administrative business plans
of routine medical services. Most of these institutions are already equipped with
VS scanners but none of them are used in routine diagnosis.

Discussions on VS image quality, or accuracy of VM based diagnosis, play only a minor
role if any at all. All serious reports of telepathology and VS experiences display
congruent evidence that VM is at least an equal if not superior diagnostic tool compared
to conventional microscopy [1,6,8]. Diagnoses can be evaluated from VS without legal problems to my knowledge.

Experiences of VM applied in routine diagnosis have been reported from one private
Institute of Pathology located in Heerlen, The Netherlands [8]. It started with VS based routine diagnosis in 2006; and then an additional University
Institute located in Uppsala, Sweden followed. They reported no difficulties in performance
or increased diagnostic failures.

Some additional (minor) constraints that might influence the implementation of VM
into routine tissue-based diagnosis include:

1. The new laboratory and LIS configuration have to be approved by a new third party
certification, which is again a question of investment.

2. Glass slides have to be stored in VM as usual, in Germany this is for mandatory
for at least 10 years. Herein, VM produces additional costs, and does not save money.

3. VM implementation only adds a link to the chain of laboratory workflow. It can
only be used for detailed workflow monitoring at its end stage, i.e. does not lower
the costs of laboratory certification.

4. VM is an appropriate tool to fasten and improve communication between the laboratory
and pathologist. This is only of importance in large institutions.

5. VM opens the door to communicative medicine. Specific medical information obtained
from different sources can be included into VM without substantial extra effort. Again,
large institutions will receive the highest benefit of this.

6. VS scanners are only a tool and are open to human error, in the same sense that
a car is. The car does not require specific administrative regulations; wrong driving
is the fault of the driver and not of the car.

7. The scanned VS could be subject to FDA approval or other inspections, although
they are viewed continuously by pathologists. The scanner itself is of no harm, and
any dysfunction will be immediately noticed, especially in routine diagnostics.

In conclusion, the recognition of the advantages of VM by the appropriate administration
requires some intelligence. The idea of the implementation of VM is an unusual consideration,
however, if performed, an action which in German would be called “nachhaltig” (its
English equivalent roughly translates to “especially good in future taking into account
less momentary success”; or to be less precise, “to achieve sustained success”). Most
administrations currently seem to be unable to perform or permit a “nachhaltige” action,
neither in Germany nor elsewhere in Europe.

However, there remains another option which gives us hope. This is the enormous amount
of money that is currently invested by the industry, which hints at future reimbursement
by appropriate administrative actions. Fortunately, no proven scientific or other
no financial constraints against this action seem to exist as far as I know.